Selective transmission of R5 HIV-1 variants, gatekeeping X4 HIV-1. To enter target cells, HIV-1 uses CD4 and a coreceptor that in vivo is either CCR5 (for R5 HIV-variants) or CXCR4 (for X4 HIV-1 variants). Although both R5 and X4 HIV-1 variants are present in body fluids (semen, blood, cervico-vaginal and rectal secretions), almost exclusively, R5 HIV-1 appears to transmit infection and dominates the early stages of HIV disease. Moreover, recent sequence analysis showed that a single virus initiates the infection in the majority of cases. Therefore, there is a gatekeeper that selects R5 over X4 HIV-1 and may operate among R5 HIV-1 variants to select a single variant. Careful analysis of such potential gatekeeping mechanisms associated with each of the transmission routes shows that they are too imperfect to explain the almost perfect selection of R5 over X4 in HIV transmission. We suggest that instead of one strong gatekeeper there are many weak ones, whose superimposition is sufficient to protect against X4 HIV-1 infection and potentially select transmittable variants from a pool of R5 HIV-1 variants. In conclusion, we think that the principle of multiple barriers is more general and not restricted to the protection against X4 HIV-1, but can be applied to other phenomena when one factor has a selective advantage over the other(s). In the case of gatekeepers for HIV-1 transmission, the task is to identify these factors and to decipher their molecular mechanisms. The knowledge of the gatekeepers localization and function may enable us to facilitate existing barriers against R5 transmission and to erect new ones against all HIV-1 variants. In general, understanding the gatekeeping mechanisms of HIV-1 transmission is critically important for the development of effective HIV-1-preventive measures (microbicides, virustats, and vaccines). Experimental study of the early barriers in HIV-1 transmission to cervico-vaginal tissue ex vivo As the cervico-vaginal tissue is the first gateway for HIV-1 during heterosexual transmission, the female lower genital tract may serve as the first gatekeeper, conferring protection against at least some HIV-1 variants. Using surgical samples of cervico-vaginal tissue (available commercially), we developed an ex vivo system to study HIV male-to-female vaginal transmission, and investigated the gatekeeping mechanisms in this system. Understanding these mechanisms requires a robust knowledge of the phenotypes, and of the functional features of the cells in the female lower genital mucosa, which are implicated in the early events of HIV-1 transmission. Such knowledge was lacking in the published literature. Using an original flow-cytometry protocol, we found that in the cervico-vaginal tissue, CD4 and CD8 T cells are predominantly of the highly differentiated effector memory phenotype with a variable pattern of expression of activation markers. The high prevalence of effector memory cells among cervico-vaginal T cells may be critical for the highly efficient defense against various incoming pathogens. We identified the phenotype, as well as the activation and differentiation status, of CD4 T cells that are preferentially infected by HIV-1 that is transmitted to cervico-vaginal tissue ex vivo. Preferential infection of activated CD38+ CD4 T cells was followed by a similar activation of HIV-1-uninfected bystander cells, creating a vicious circle between R5 HIV-1 replication and the number of cell targets that support this infection. These results provide new insights into the dependence of HIV-1 infection and the dissemination of the activation of cervico-vaginal lymphocytes. Remarkably, isolated blocks of cervico-vaginal tissue retain an in vivo gatekeeping mechanism that selects between R5 and X4 HIV-1 infection in spite of the broad expression of both CXCR4 and CCR5, the co receptors for X4 and R5 HV-1. We attributed the higher susceptibility of cervico-vaginal tissue to R5 HIV-1 infection over X4 HIV-1 infection, to the low abundance of the CD27+ CD28+ effector memory CD4 T cells. In the system of cervico-vaginal tissue ex vivo, we are now investigating potential barriers that select for the R5 HIV-1 virions that transmit infection through the mucosal barriers. Determinants of protective barriers among high-risk seronegative persons Theoretical and experimental laboratory evidences of the existence of multiple gatekeeping mechanisms need to be translated into clinical research. Growing epidemiological literature indicates that there are persons who have been exposed to HIV infection but who have remained uninfected. We suggest launching a complex study to investigate the natural barriers of resistance to HIV infection by using a cohort of uninfected persons where the risks for infection are thought to be high. In addition to the 32 base pair deletion in the CCR5 gene which, at the date of this report, is the only mechanism associated with a high protection level from HIV infection, these cohorts will allow the investigation of alternate mechanisms of protection. According to our concept a number of imperfect gatekeepers contribute to the protection from infection. Moreover, this protection may not be consistent over time, and may be affected by various factors such as hormonal environment, innate and adaptive immunity as well as the individuals microbiome so that an individual apparently protected for one period of high-risk exposure, may lose this relative protection at other times. A consortium of laboratories including our Section has been established to identify natural gatekeepers in vivo.